Roles of intralesional bacteria in the initiation and progression of oral squamous cell carcinoma

Abstract Background Oral squamous cell carcinoma (OSCC) is the predominant form of head and neck cancer, often diagnosed at late stages, resulting in a poor prognosis. Recent studies indicate a potential association between OSCC and microbial presence. Microorganisms have been identified in various tumors and lesions, including OSCC and oral potentially malignant disorders (OPMDs). Intralesional microbiota are considered important components of the tumor microenvironment (TME) and may contribute to carcinogenesis. Methods Sources were collected through thorough searches of databases PubMed and Embase. The review focused on microbial characteristics, potential origins, and their impact on cancer progression. Results Bacteria display varying abundance and diversity throughout the stages of OSCC and OPMDs. Intraleisional bacteria may have diverse sources, including not only oral plaque and saliva but also potentially the gut. Intralesional bacteria have both pro‐carcinogenic and anti‐carcinogenic effects, affecting processes like cell proliferation, invasion, and immune response. Conclusions Intralesional microbiota are crucial in OSCC and OPMDs, influencing both disease progression and treatments. Despite their significance, challenges like inconsistent sampling and microbial identification remain. Future research is required to fully understand their role and improve clinical applications.

and betel nut chewing. 4,5However, the etiology of OSCC remains incompletely understood.In recent years, numerous studies have suggested the association between microbiota and oral cancer.
The human microbiota is a comprehensive term that includes all microorganisms found on and within the human body. 6Imbalances of the human microbiota can cause or exacerbate various diseases, such as inflammatory bowel disease (IBD), autoimmune disorders, and even cancer. 7,8Cancer arises from a combination of genetic and environmental factors, and approximately one-fifth of which can be associated with the human microbiome. 9n contrast to the oral and gut microbiota, intralesional microbiota has received less attention in the past due to its minimal biomass. 10In recent years, the rapid development of sequencing technology has brought intralesional microbiota under the spotlight. 11Microbiota have been extensively confirmed to exist in various types of tumors, with distinct microbial compositions. 12][15] Oral cavity is the second-largest microbiota in the body after the gut, with over 700 identified bacterial species. 16merging evidence has verified the existence of intralesional microbiota in OPMDs and OSCC, suggesting a potential impact on these disorders. 17,18However, previous reviews on microbiota and OSCC mostly relied on surface samples, such as saliva and swabs.Microbial communities in surface samples are unstable and influenced by factors like oral hygiene, tobacco use, and diet. 19It is important to note that there are significant differences in microbial compositions and community structures between surface and tissue samples. 20,21ccording to reports, bacteria constitute the majority of the intralesional microbiota, while fungi and viruses constitute a smaller proportion. 18,22Therefore, this review aims to summarize the characteristics of intralesional bacteria, including representative bacterial alterations in OPMDs and OSCC, the possible origins of these bacteria, and their potential roles and mechanisms in the initiation and progression of OSCC.

| DYNAMIC ALTERATIONS OF INTRALESIONAL BACTERIA IN OPMDs AND OSCC
There is a significant correlation between bacterial community structure and the progression of diseases.Diversity variations reflect overall bacterial changes.4][25][26] Particularly, studies indicated the decrease of bacterial diversity during the progression from PVL to OSCC, and the significant loss of diversity in advanced T4 stage OSCC compared to the early stage. 18,27,28Changes in biodiversity may be attributed to the increased growth of potential pathogens during disease progression.Additionally, to clarify the specific bacterial compositions within OPMDs and OSCC lesions, the 16S rRNA gene sequencing results are summarized, as shown in Table 1.][31] Researches regarding the bacterial compositions of OPMDs tissues are still lacking.A study involving various OPMDs revealed a significant increase in the relative abundance of Fusobacterium nucleatum (F.nucleatum) and a decrease in Streptococcus species, compared to the healthy mucosa. 25PVL is considered a severe subtype of OLK and exhibits the highest malignant transformation rate among OPMDs. 3PVL showed a loss of diversity and enrichment of pathogens such as Oribacterium sp.oral taxon 108 and Campylobacter jejuni. 27As PVL progressed to OSCC, the enrichment of Lachnospiraceae, Selenomonas sputigena, etc. was observed. 18Additionally, another study indicated the depletion of Streptococcus and Rothia during the oral carinogenesis of OPMDs. 28he trends of commonly identified bacteria in OSCC are analyzed using a heatmap, as shown in Figures 1 and  2. At the phylum level, the abundances of Fusobacteria and Campylobacterota significantly increased, whereas Actinobacteriota significantly decreased.However, the reported trends of changes in 4 phyla in OSCC lesions show inconsistencies 18,21,23,24,28,[32][33][34][35][36][37] (Figure 1).At the genus level, 8 genera, including Fusobacterium, significantly increased in OSCC, while 5 genera including Veillonella and Stenotrophomonas significantly decreased.Changes of 5 genera show inconsistencies in OSCC 4,[18][19][20][21]23,[26][27][28][32][33][34][35][36][37][38][39][40] (Figure 2). Specially, a hher level of F. nucleatum in OSCC has been widely reported.25,33,[41][42][43] Moreover, bacteria show specific compositions across different stages of OSCC.In the early stages, genera Clostridiales, Desulfovibrionaceae, and Leptotrichia are enriched, while Pedobacter is enriched in the late stage.28 From the early to late stage, the relative abundance of Solobacterium moorei is positively correlated with OSCC progression.21 The prevalence of Porphyromonas gingivalis (P. gingivalis)was found to be elevated in patients with T3-T4 stage OSCC, poorly differentiated subtypes, and lymph node metastasis.41 In addition, phyla Firmicutes, Actinobacteria, and Fusobacteria-related taxa exhibit notable predictive accuracy in diagnosing or prognosing OSCC.34 Increased levels of Capnocytophaga and reduced levels of Streptococcus could potentially act as biomarkers for advanced OSCC.Traditional 16S rRNA gene sequencing is limited to genus-level microbial community classification and does not support accurate species-level identification.Future studies may leverage more advanced sequencing methods like 5R 16S rRNA gene sequencing and 2bRAD sequencing to explore the microbial composition in tissues with low microbial load, enabling precise genus-level results in the investigation of OPMDs and OSCC lesions.12,44 3 | POTENTIAL ORIGINS OF THE

INTRALESIONAL MICROBIOTA
The current research has mentioned the potential origins of intralesional microbiota, particularly emphasizing the association with periodontitis.Periodontal pathogens, especially P. gingivalis, dominate the entire periodontitis-associated OSCC process. 29Periodontal pathogens are enriched in cancerous tissue, with bacterial distributions similar to those in subgingival plaque.The relative abundance of the key components, F. nucleatum and P. gingivalis, is significantly positively correlated with that in subgingival plaque. 41Streptococcus anginosus (S. anginosus) shares the same genotype in OSCC tissue and dental plaque. 45Another research revealed identical P. gingivalis nucleotide sequences in OSCC tissue and saliva, along with the consistent distribution of fimA genotypes in both, suggesting that P. gingivalis in OSCC may originate from the salivary microbial reservoir. 46Microbial compositions in the lesions are similar to those in adjacent normal tissues, which suggest that intralesional bacteria might originate from nearby tissues. 35,41In OLP, chronic inflammation may alter epithelial permeability and disrupt the epithelial barrier, allowing bacteria to penetrate the epithelial and lamina propria. 19Specifically, certain gut-specific bacteria were also detected in OSCC, such as Clostridium neonatale. 41This indicates intraleisional bacteria may have sources other than the oral cavity, such as the intestines or other anatomical sites.
With the knowledge of the intralesional microbiotas and related studies, it can be inferred that microorganisms within OSCC and OPMDs tissues have multiple sources.Revealing the sources of intralesional microbiota helps understand the mechanisms involved in OPMDs and OSCC, and provides inspiration for disease prevention and treatment.

INTRALESIONAL BACTERIA IN OSCC INITIATION AND PROGRESSION
Polymorphic microbes have been considered an emerging hallmark of cancer. 47As a crucial component of the tumor microenvironment (TME), microbiotas play complex roles in cancer development.Intralesional bacteria can contribute to carcinogenesis of normal cells or exacerbate existing OSCC via various mechanisms, including promoting cell proliferation, inhibiting apoptosis, and inducing chronic inflammation and immune suppression.

| Promote cell proliferation
Continuous cell proliferation is a critical characteristic of cancer.Normal cells maintain cellular homeostasis by finely regulating growth signals. 48However, the presence of intralesional bacteria disturbs this balance and accelerates the proliferation of epithelial cells or cancer cells. 49roteomic analyses have revealed that P. gingivalis leads to a significant upregulation in the levels of cyclins and cyclin-dependent kinases (cdks) in oral epithelial cells, which accelerate transitions between different phases of the cell cycle. 50,51Exposure to P. gingivalis or F. nucleatum activates toll-like receptors (TLRs)/interleukin-6 (IL-6)/ signal transducer and activator of transcription 3 (STAT3) axis in human oral keratinocytes (HOK), and induces cyclin D1 expression. 52In a specific environment containing ethanol, the interaction between Streptococcus and human papillomavirus (HPV) causes malignant phenotypes in HOK, including uncontrolled cell proliferation. 53n OSCC, bacteria can relieve the restrictions on cell proliferation through multiple mechanisms.P. gingivalis accelerate G1 and S phases of cell cycle also via the upregulation of cyclin D1 through the miR-21 (microR-NA-21)/PDCD4 (Programmed Cell Death Protein 4)/ AP-1 (Activator Protein-1) negative feedback signaling pathway. 54Similarly, MYC is a crucial driver gene that accelerates the cell cycle.In OSCC, Cutibacterium acnes is strongly associated with the enriched MYC pathway. 55P53 is another crucial tumor suppressor protein that regulates cell cycle progression, DNA repair, and apoptosis.F. nucleatum enhances the proliferation ability of tongue squamous cell carcinoma cells by causing DNA damage via the downregulation of Ku70/P53 pathway. 56Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor, which plays a crucial role in cellular processes including proliferation, migration and differentiation. 57Human defensins may serve as one of the ligands of EGFR.P. gingivalis infection upregulates human defensins expression and activates downstream EGFR signaling, thus promoting cell proliferation. 58Enterococcus faecalis produces hydrogen peroxide when interacting with host cancer cells.Hydrogen peroxide promotes EGFR activation, which contributes to proliferation potentially via mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MAP2K/ERK). 59Treponema denticola can invade Cal-27 cells, directly promoting cell proliferation and inhibiting apoptosis via transforming growth factor-beta (TGF-β) pathway. 60Additionally, OSCC cells infected with bacteria show activated signaling pathways related to cell proliferation, including janus kinase 1/signal transducer and activator of transcription 3 (JAK1/STAT3), nuclear factor kappa B (NF-κB), and phosphoinositide 3-kinase (PI3K) 52,61,62 (Figure 3A).

| Inhibit cell death
Apoptosis is a form of programmed cell death, serving as a natural defense againist cancer progression.Evasion of cell death enables cells to prolong their survival and accumulate more mutations. 63Studies have shown that both epithelial cells and OSCC cells experience various antiapoptotic effects when co-cultivated with bacteria.
P. gingivalis in gingival epithelial cells upregulates the expression of miR-203, resulting in the reduction of downstream targets such as the suppressor of cytokine signaling 3 (SOCS3) mRNA.The decreased SOCS3 level downregulates apoptosis through JAK/STAT3 pathways. 64The B-cell lymphoma-2 (Bcl-2) family comprises both anti-apoptotic and pro-apoptotic members, whose counterbalancing regulates the apoptotic trigger. 48A meta-analysis revealed a significant upregulation of genes suppressing apoptosis in epithelial cells infected with P. gingivalis, including the anti-apoptotic protein Bcl-2. 65Disruption of normal adhesion between cells and the extracellular matrix (ECM) triggers another programmed cell death response called anoikis.P. gingivalis enables oral keratinocytes to develop anoikis resistance potentially through the Bcl-2 protein. 66. gingivalis ecto-nucleoside diphosphate kinase (NDK) is an extracellular adenosine 5′-triphosphate (ATP) enzyme.NDK can hydrolyse extracellular ATP, thereby inhibiting ATP-dependent apoptosis mediated by purinergic receptor P2X (7).67 Furthermore, a separate study demonstrated that NDK inhibits apoptosis by phosphorylating Heat-shock-protein-27 (Hsp27) in gingival epithelial cells.Phosphorylation of Hsp27 deactivates the proapoptotic Bcl-2-associated X protein (Bax) and suppresses the release of cytochrome c. 68 P. gingivalis also inhibits cell apoptosis partly by controlling the intrinsic mitochondria cell death through a caspase-dependent apoptotic pathway.65 In OSCC cells, studies revealed that the activation of TLR2 by bacteria leads to the upregulation of miR-146a-5p and subsequent suppression of caspase recruitment domain-containing protein 10 (CARD10), which facilitates resistance to cell death.69 T. denticola inhibits apoptosis of cancer cells through TGF-β pathway.60 T. denticola and its toxin Treponema denticola chymotrypsin-like proteinase (Td-CTLP) are significantly associated with higher expression of TLR7 and TLR9.The activation of mitogenactivated protein kinase (MAPK) signaling via TLR7 and TLR9 significantly promotes cell survival 70 (Figure 3B).

| Deregulate cell metabolism
By disturbing cell metabolism, carcinogenic bacteria may create conditions conducive to the advancement of OSCC.NDK of P. gingivalis can hydrolyse extracellular ATP, which influences associated metabolic processes in oral epithelial cells. 67Stimulation of P. gingivalis W83 membrane components triggers a strong metabolic gene expression response in OSCC cells. 71Hypoxia-inducible factor 1α (HIF-1α) regulates cellular responses in low-oxygen environments, affecting transcription, angiogenesis, and metabolism.OSCC cells show strong HIF-1α expression in the cytoplasm, especially in areas densely populated by F. nucleatum. 33Glucose transporter (GLUT) is a key transmembrane protein family essential for cellular glucose transport and metabolism.Intratumoral F. nucleatum facilitates the aggregation of GLUT1 on the cell membrane through the N-acetylgalactosamine (GalNAc)/autophagy/TBC1D5 axis, leading to an increased level of glucose glycolysis and lactate production. 17Excessive iron and vitamin K2 exhibit potential anticancer effects.In OPMDs and OSCCs, microbiomes take up extracellular iron and vitamin K2, thereby restricting the utilization by tumor cells.In this way, microbiomes adjust iron and vitamin K2 to suitable levels for cancer progression. 72Furthermore, it has been validated in 4-nitroquinoline-1-oxide (4-NQO)-induced OSCC models that P. gingivalis alters the metabolism of free fatty acids (FFA) in tongue tissues by upregulating fatty acid synthase (FASN) and acetyl-CoA carboxylase 1 (ACC1) via the de novo fatty acid (FA) synthesis pathway 73 (Figure 3C).

| Activate invasion and metastasis
OSCC originates from the oral epithelium.During carcinogenesis, cell morphology changes, and adhesion between cells and the ECM decreases. 48pithelial cadherin (E-cadherin) regulates calciumdependent cell adhesion in tight junctions, contributing to the cellular structural integrity and stability.E-cadherin is a well-established tumor suppressor, particularly in preventing cell invasion and migration. 74Studies have demonstrated the downregulation of E-cadherin in oral epithelial cells and cancer cells infected with bacteria.6][77][78] In immortalized oral keratinocytes, P. gingivalis develops anoikis resistance to sustain invasion.In this process, predominant E-cadherin expression shifts to neural cadherin (N-cadherin), which promotes cell detachment and metastasis. 66Protein claudin-4 is also crucial to the formation of tight junction.In OSCC cells, exposure to Clostridium perfringens' enterotoxin (CPE) leads to the nuclear translocation of claudin-4, reducing intercellular adhesion and promoting invasion and migration. 79Matrix Metalloproteinases (MMPs) are a family of zinc-dependent enzymes that degrade components of the ECM, particularly the basement membrane.When cells lose the constraints of the basement membrane, their activity and invasiveness increases. 80In OSCC cells infected with P. gingivalis, the overexpression of proMMP9 is stimulated through the activation of proteinase-activated receptor-4 (PAR-4) and downstream signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2), p38, or NF-κB.Subsequently, the proenzyme is activated in a P. gingivalis gingipain proteases-dependent way to exert the aforementioned functions. 81,82Additionally, an upregulation of MMP1, MMP2, MMP7, MMP10 were also observed in cells exposed to bacteria. 78,83pithelial-mesenchymal transition (EMT) is a reversible transformation in which epithelial cells acquire the traits of mesenchymal cells, including mobility, stemness, and invasiveness. 48Long-term infection by P. gingivalis induces EMT phenotype in primary oral epithelial cells (OECs). 78n gingival epithelial cells, fimA-positive P. gingivalis activates zinc finger E-box binding homeobox 1 (ZEB1) and its nuclear translocation, potentially initiating a stable partial EMT process. 84Similarly, bacteria promote EMT in OSCC.Studies have reported that periodontal pathogens P. gingivalis and F. nucleatum upregulate EMT-related genes, including EGF, TGF-β1, Twist and SNAI1 in OSCC cells. 62,76,78,85P. gingivalis and F. nucleatum also promote cell invasion and migration via activation of integrin/focal adhesion kinase (FAK) signaling, triggered by TLR/myeloid differentiation primary response 88 (MyD88). 86F. nucleatum may initiate the EMT through the lncRNA MIR4435-2HG/miR-296-5p/ protein kinase B (Akt2)/SNAI1 pathway in cancer cells. 87. gingivalis activates JAK1/STAT3 signaling via chemokine (C-X-C motif) ligand 2/receptor 2 (CXCL2/CXCR2) axis to promote EMT process. 88Furthermore, other signaling pathways such as wingless-related integration site/nuclear factor of activated T-cells (Wnt/NFAT) and Notch were also reported to play a role in EMT in OSCC cells infected with bacteria 43,62,88,89 (Figure 3D).

| Induce genome instability and mutation
Microbial genotoxicity induces DNA damage in host cells.As DNA damage intensifies with cell proliferation, the accumulation of gene mutations promotes cancer development. 48Periodontal pathogens arrest cell cycle at the S phase, activate upstream signals of ataxia telangiectasia and Rad3-related protein-checkpoint kinase 1 (ATR-CHK1), and inhibit the activation of CHK1, thus triggering DNA damage. 90Additionally, study confirmed DNA double-strand breaks in OSCC cells infected with F. nucleatum, mediated by the reduction in Ku70 and P53 expression 56 (Figure 3E).

| Stimulate vasculature
Angiogenesis influences cancer advancement by ensuring a vital nutrient supply to tumors and fostering their continual expansion.Vascular Endothelial Growth Factor (VEGF) is a pivotal signaling protein that regulates angiogenesis.Numerous studies have indicated the production of interleukin-6 (IL-6) after the stimulation of bacteria, which subsequently enhances the expression of VEGF in OSCC cells. 52,77,91Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria, commonly regarded as a potent endotoxin.LPS of P. gingivalis promotes the generation of VEGF2 within the gingival epithelial cells, leading to the initiation of blood vessel formation 92 (Figure 3F).

| Promote chronic inflammation
Bacteria promote chronic inflammation directly or indirectly, thereby creating a friendly environment for the progression of OPMDs or OSCC. 93Studies have revealed that intralesional microorganisms can activate pro-inflammatory immune responses both in epithelial cells and OSCC cells, including TLR, STAT, NF-κB and MAPK signaling pathways. 49,52,62OLP is a chronic mucocutaneous inflammatory condition among OPMDs, characterized by a dense T-cell infiltrate in the lamina propria.Bacteria have been observed to colonize epithelial cells and infiltrate T cells, while the expressions of T cell chemokines CXCL10 and chemokine (C-C motif) ligand 5 (CCL5) increased.Intracellar bacteria may act as target antigens for T cells to promote basal cell liquefaction and barrier impairment, thus contributing to chronic inflammation. 32Prevotella melaninogenica promotes IL-36γ expression via the activation of transient receptor potential vanilloid receptor-1 (TRPV1) in OLP. 94Stimulated by P. gingivalis LPS, fibroblasts in OLP lesions undergo transformation into myofibroblasts and secrete proinflammatory cytokines, including IL-6, IL-8, and tumor necrosis factor-alpha (TNF-α). 95Additionally, P. gingivalis LPS promotes CCL2 and chemokine (C-C motif) receptor 2 (CCR2) expressions through TLR-4/NF-κB pathway in OLP. 96Fusobacteria LPS stimulates TLR4 and induces the production of IL-8 in cancer cells. 62acteria also indirectly promote inflammation through the secretion of virulence factors.Gingipains are cysteine proteases produced by P. gingivalis, consisting of two types: Arg-gingipains and Lys-gingipains.Arg-gingipains can increase inflammatory cytokine expression in oral keratinocytes through the selective cleavage of PAR-1. 97treptolysin S (SLS) is a toxin secreted by the βhemolytic S. anginosus subsp.anginosus (β-SAA).In OSCC cells, SLS facilitates Ca2+ influx, thus increasing the expression of IL-6 and IL-8 98 (Figure 4).

| Promote immunosuppression
In oral carcinogenesis, there is an enrichment of immunesuppressive cells and molecules within the microenvironment. 99The TME plays a potential role in creating such a tumor-friendly condition by selectively enriching the non-immunogenic or immune-suppressive bacteria. 28 spatial analysis revealed that the distribution of bacteria in OSCC is highly-organized rather than random.Intratumoral bacterial communities often colonize niches with reduced vascularity and strong immunosuppression.This organization facilitates cancer progression via regulation of immune cells and epithelial cells. 85ertain bacteria, including Rothia and Streptococcus, have a positive correlation with naive and central memory T cells.However, their abundance decreases significantly in the TME, suggesting the diminishing antitumor adaptive immune response in OSCC.Additionally, bacteria such as Corynebacterium and Prevotella are significantly associated with the expression of GATA3 (a master regulator of T helper 2-cell differentiation) and IL-10, which mainly contribute to immune suppression. 28ntratumoral F. nucleatum can reduce the density of the infiltrated T cells and induce their dysfunction, thereby inhibiting T-cell immunity. 93Moreover, bacteria inhibit immune responses through established inhibitory receptors.Exposure to Fusobacterium and P. gingivalis upregulates the level of programmed death ligand 1 (PD-L1).The increased PD-L1 serves as a mechanism to evade the cytotoxic responses of T cells, potentially via a receptorinteracting protein kinase 2 (RIP2)-mediated process. 38,100. nucleatum interacts with receptor T cell immunoglobulin and ITIM domain (TIGIT) through Fap2 protein to suppress the immune responses of Natural killer (NK) cells and T cells against OSCC cells.101 In addition to directly influencing adaptive immunity, bacteria also influence components of the innate immune system.Myeloid-derived suppressor cells (MDSCs) exert a potent immunosuppressive ability.In 4-NQO-induced OPMDs and OSCC mice, P. gingivalis activates the expression of CCL2, CXCL2, IL-6 and IL-8 to recruit MDSCs and infiltrate lesions.102 M2-type tumor-associated macrophages (M2-TAMs) are a crucial contributor to the inhibition of immune responses in the TME. Perdontal pathogens significantly promote the M2 polarization of macrophages when co-cultivated with OSCC cells.103 In addition, P. gingivalis was observed to shield cancer cells from macrophage phagocytosis.104 Regarding the underlying mechanism, P. gingivalis may facilitate the M2 polarization process through a key modulator downstream of kinase 3 (DOK3) via TNF and MAPK signaling pathways.105 In 4-NQO-Induced OSCC mice, intratumoral F. nucleatum promotes the formation of M2-TAMs by upregulating lactate expression through GLUT1.17 Likewise, tumor-associated neutrophils-2 (TANs-2) exhibit protumor effects by suppressing immune responses and facilitating proliferation.106 Studies have revealed that P. gingivalis recruits TANs-2 via the activation of CXCL2/ CXCR2 axis in the TME of OSCC 88 (Figure 4).

| Anti-cancer effects
Apart from the pro-cancer effects, some bacteria also play a role in OSCC regression.Periodontitis negativeassociated bacteria (PNB), including Neisseria sicca and Corynebacterium matruchotii, have the potential to exert anticancer effects.PNB significantly downregulate the level of IL-6, IL-8, MMP-9 and cyclin D1, thereby suppressing the proliferation, migration, and invasiveness The reported trend of abundance changes of bacteria in OSCC or OPMDs at the genus level. of OSCC cells. 107In addition, PNB enhance genome stability by activating DNA repair pathways and promoting pyroptosis mediated by the inflammasome containing domains of protein 3 (NLRP3). 108Veillonella parvula and its major metabolite, sodium propionate (SP), inhibit abnormal proliferation and facilitate apoptosis by blocking the tumor-associated calcium signal transducer 2 (TROP2)/phosphoinositide 3-kinase kinase B (Akt) pathway. 109Lactobacillus plantarum is an important bacterium that commensally lives in the oral | 13 of 19 LUO et al. cavity.This bacterium induces apoptosis of oral cancer cells through the upregulation of phosphatase and tensin homolog (PTEN) and downregulation of MAPK. 110dditionally, multiple studies have reported a higher relative abundance of the genus Streptococcus in normal tissues compared to OSCC or OPMDs, suggesting its potential anticancer effect. 28The supernatant of S. anginosus was observed to promote apoptosis and reduce proliferation and invasion in OSCC cells. 111Streptococcus cristatus decreases the expression of pro-inflammatory cytokines by influencing NF-κB signaling pathways. 112. gingivalis is relatively special.Despite the majority of studies indicating its tumorigenic effects, there are still researches reporting its anticancer properties and the association with the longer survival in OSCC patients. 46 recent study has shown that P. gingivalis overturned the immunosuppressive TME via the downregulation of mucin 1 (MUC1). 113Phosphoethanolamine dihydroceramide (PEDHC) derived from P. gingivalis decreases the level of acid ceramidase and promotes the aggregation of ceramide, thereby accelerating cancer cell death. 114nother study reported that P. gingivalis induces autophagy and inhibits proliferation of oral cancer cell by downregulating cyclin D1 and cdk4. 115These results seem to be contradictory to the other studies.The discrepancy may be attributed to varied status of P. gingivalis during cell infection.Heat-killed P. gingivalis shows reduced invasiveness and apoptotic effects compared to live strain. 104,116In addition, multiple factors such as cell types, cell status, and duration of treatment also affect the outcomes. 115More evidence is needed for a thorough insight of the exact effect of P. gingivalis (Figure 5).

| DISCUSSION
OSCC is characterized by high aggressiveness and metastasis, with limited therapeutic options. 117Although substantial progress has been achieved in the past decades, patients with advanced-stage OSCC still face an undesirable prognosis. 118This paper provides an overview of the intralesional bacteria in OSCC.Bacteria exhibit varying diversity and composition in different diseases or phases.Particularly, specific bacteria are enriched or reduced at specific stages, such as Streptococcus, Rothia which are enriched in normal controls but depleted as OSCC progress.This dynamic change provides important clues to the role of bacteria in disease development.The significantly increased bacteria can be potential pathogens that contribute to or adapt to the TME, whereas those declining are more likely part of micro-ecological balance.Currently, little is known about the exact sources of intralesional microbiota.Understanding of their origins and migration helps identify key bacteria and deepens the comprehension of the microbial mechanisms in diseases.
In terms of the potential roles in OSCC initiation and progression, intralesional bacteria manifest dual nature, with both pro-carcinogenic and anti-carcinogenic effects.Representative pro-carcinogenic bacteria, including F. nucleatum, P. gingivalis and T. denticola, promote the occurrence and development of OSCC through multiple mechanisms, particularly by enhancing proliferation, invasion, inflammation, inhibiting apoptosis, and creating an immunosuppressive microenvironment.On the contrary, anti-carcinogenic bacteria, notably N. sicca and C. matruchotii, act as probiotics to prevent the OSCC F I G U R E 3 Mechanisms of intralesional microbiotas promoting the initiation and progression of OSCC.(A) Promoting cell proliferation.Intralesional bacteria, such as P. gingivalis, F. nucleatum, and Enterococcus faecalis, can directly promote cell proliferation or release toxins that facilitate this process.Through the activation of membrane receptors, such as toll-like receptors (TLRs), the NF-κB, MAPK, and STAT3 pathways are stimulated.The downstream cyclins and the MYC gene expression upregulate, which accelerates the cell cycle and promotes cell proliferation.The upregulation of human defensins triggered by P. gingivalis and the production of H 2 O 2 triggered by E. faecalis can both activate Epidermal Growth Factor Receptor (EGFR), thereby promoting cell proliferation through pathways including MEK/ERK.(B) Inhibiting cell death.Microbiotas participate in inhibiting cell death through pathways such as JAK/STAT3 and PI3K.P. gingivalis ecto-nucleoside diphosphate kinase (NDK) depletes extracellular ATP to reduce ATP-dependent apoptosis mediated by P2X( 7) receptor (P2X(7)R).P. gingivalis also induce resistance of host cells to anoikis, a process where loss of cell adhesion to the extracellular matrix (ECM) triggers cell death.(C) Deregulating cellular metabolism.Intralesional bacteria consume extracellular iron and vitamin K2, thus reducing the potential anticancer effects of these nutrients.Infection with P. gingivalis causes abnormal fatty acid metabolism in OSCC cells, resulting in elevated levels of free fatty acids (FFA).F. nucleatum upregulates the expression of glucose transporter 1 (GLUT1) and HIF-1α in OSCC cells, which influences the metabolic processes involved in glucose metabolism and responses to hypoxia.(D) Activating invasion and metastasis.The increase in cytokines such as MMPs and the decrease in cell adhesion-related proteins E-cadherin and claudin-4 promote loss of cell polarity and depleted cell-cell and cell-ECM adhesion.Cytokines released by OSCC cells and epithelial cells facilitate OSCC EMT process through multiple pathways including Wnt/NFAT1, Notch, and JAK/STAT1.Subsequently, transformed cells are able to migrate through intercellular spaces, penetrate ECM, and infiltrate surrounding tissues or enter circulation to achieve distant metastasis (Adapted from "Blood Vessel (Straight, Light Background)", by BioRe nder.com (2024).Retrieved from https:// app.biore nder.com/ biore nder-templ ates).(E) Inducing genome instability and mutation.Intralesional bacteria cause DNA double-strand breaks by suppressing the Ku70/P53 pathway.(F) Stimulating vasculature.Bacteria promote angiogenesis by stimulating vascular endothelial growth factor (VEGF) 2 production and promoting VEGF through the induction of IL-6.(Image created with BioRe nder.com, with permission).progression.P. gingivalis has been reported to exhibit conflicting pro-cancer and anti-cancer effects, which deserves further validation.
Most conclusions rely on 16S rRNA gene sequencing and co-culture experiments.However, without standardized sampling criteria and laboratory procedures, the comparability of conclusions across different studies may be questionable.In vitro experiments alone cannot entirely replicate the intricate TME and the crosstalk among cells in vivo; co-culture with individual bacteria also overlooks communications between bacteria; different types of cancer cells may exhibit significant variations in responses to the same bacteria.In addition, the 16S rRNA gene sequencing might encounter challenges when processing samples with low microbial biomass.The limited precision makes it difficult to accurately identify microbial community structures at the species level.Therefore, further research employing new techniques such as 2bRAD is necessary to reveal the biodiversity and variations in gene expression more comprehensively.
Taken together, the intralesional microbiome is a critical component of the pathological microenvironment, which exerts a multifaceted influence on disease progression.There are still many issues to be addressed in this field, requiring technological advancements and concerted efforts.Exploration of intralesional microbiota has considerable clinical significance, which may bring fresh insights into cancer treatment.

FUNDING INFORMATION
This work was supported by grants from the National Natural Science Foundations of China (No. 82370968) and the Natural Science Foundation of Chongqing (No. CSTB2022NSCQ-MSX1148).The funding agencies had no role in the study design, collection, analysis, or interpretation of data, writing of the report, or the decision to submit the article for publication.

F I G U R E 1
The reported trend of abundance changes of bacteria in OSCC or OPMDs at the phylum level.

F I G U R E 4
Mechanisms of intralesional bacteria promoting chronic inflammation and immune suppression.Bacteria and their toxins, including streptolysin S (SLS), peptidoglycan, gingipains, and LPS, exert significant influence on the immune microenvironment.They stimulate the secretion of pro-inflammatory cytokines and leukocyte chemokines by tumor cells and epithelial cells, such as IL-6, IL-8, MMPs, and CXCL10.OLP-associated fibroblasts, when stimulated by LPS, transition into myofibroblasts and release cytokines IL-6, IL-8, and TNF-α.Inflammatory signaling pathways such as NF-κB, MAPK, JAK/STAT are activated.The chemokines activate and recruit immune cells, including T cells, macrophages, neutrophils, and monocytes, to promote inflammation.Additionally, the level of PD-L1 in tumor cells increases to evade immune responses.Intralesional F. nucleatum inhibits the cytotoxic effect of natural killer (NK) cells and T cells by binding to their T cell immunoglobulin and ITIM domain (TIGIT) receptors through the Fap2 protein.Microbiotas upregulate the expression of TNF-α, Transforming growth factor-beta (TGF-β), CXCL2, CXCL10, and CCL2 in OSCC cells, promoting the infiltration of myeloid-derived suppressor cells (MDSCs), CD11b + cells, Treg cells, and the formation of tumor-associated neutrophils (TANs) and M2 polarization of tumor-associated macrophages (TAMs), which create an immune-suppressive environment conducive to tumor progression.(Image created with BioRe nder.com, with permission).

F I G U R E 5
The potential probiotics' anticancer effects.The supernatant of Streptococcus anginosus reduces proliferation and invasion and promotes apoptosis in cancer cells.Streptococcus cristatus decreases the expression of pro-inflammatory cytokines by influencing NF-κB signaling pathways.Neisseria sicca and Corynebacterium matruchotii significantly inhibit the inflammatory response by downregulating NF-κB and IL-6 mRNA levels.Moreover, by activating the DNA Damage Response (DDR) and NOD-like receptor protein 3 (NLRP3) inflammasomes in OSCC cells, they inhibit cell proliferation, promote gene stability, and facilitate pyroptosis (a specific form of programmed cell death mediated by the NLRP3 inflammasome).Veillonella parvula and its major metabolite inhibit proliferation, invasion and promote apoptosis by blocking the TROP2/PI3K/Akt pathway.Lactobacillus plantarum induces cell apoptosis through the upregulation of PTEN and downregulation of MAPK.(Image created with BioRe nder.com, with permission). 28

Study, year Participants (N) Average/range of age (years) Group Bacterial ateration (increase, decrease)
Characteristics of the included studies.
T A B L E 1